PROJECT SUMMARY This R01 renewal proposal is designed to fully characterize a novel mechanism relevant to the established clinical observation that skeletal muscle lipid accumulation is strongly associated with the development of insulin resistance. Our preliminary studies have identified a potentially exciting role for the transcription factor MondoA in the coordinate control of muscle fuel metabolism and insulin signaling. This discovery was unveiled by an unbiased high-throughput chemical biology screen designed to identify small molecule probes that influence downstream pathways involved in the control of myocyte neutral lipid stores. One such molecule, SBI-477, reduced human skeletal myocyte lipid stores and increased glucose uptake. Our recent results indicate that SBI-477 is a potent inhibitor of fatty acid incorporation into triglyceride (TAG) and activates insulin signaling in human skeletal myocytes and in vivo. The downstream actions of SBI-477 are attributable, at least in part, to inhibition of the transcription factor MondoA resulting in reduced expression of target genes encoding TXNIP and ARRDC4, known suppressors of insulin signaling. These results support the hypothesis that MondoA serves a key metabolic homeostatic function in muscle by controlling fuel substrate availability by regulating genes involved in myocyte lipid storage and glucose import (via insulin signaling) in accordance with nutrient availability. We propose, however, that in states of chronic caloric excess persistent activation of MondoA becomes maladaptive, contributing to a vicious cycle of cellular lipid accumulation and insulin resistance. To test these hypotheses, we will conduct studies in human skeletal myotubes in culture and in vivo in MondoA loss-of-function mice. In Aim 1, we will delineate MondoA gene targets (direct and indirect) and pathways in human skeletal myocytes using unbiased whole genome chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq. The goal of Aim 2 is to validate the function of MondoA-regulated targets and pathways in the control of skeletal myocyte glucose and lipid metabolism under conditions of varied nutrient supply and cellular energy status. In Aim 3, the metabolic actions of MondoA will be defined in vivo in muscle-specific MondoA-deficient mice under basal conditions and after long-term administration of a high-fat diet to determine whether MondoA participates in the development of muscle lipotoxicity and insulin resistance in the context of chronic nutrient overload. The long-term goal of this project is to identify novel mechanisms and targets relevant to the early-stage treatment of muscle insulin resistance in individuals at risk for developing type 2 diabetes. The mechanistic insights gained from the proposed studies should lead to new approaches for the prevention and treatment of lipotoxic diseases relevant to other tissues including nonalcoholic steatohepatitis (NASH) and heart failure in the diabetic.